Cell cycle arrest by oxaliplatin on cancer cells.

Oxaliplatin (L-OHP) is the only platinum compound to show activity in colorectal cancer. We evaluated the cytotoxicity of L-OHP on four human cancer cell lines and its influence on the cell cycle, when treated during long exposure (72 h) and different post-incubation times (24 or 72 h). We used a panel of cell lines: HT29 (colon cancer), MCF7 (breast cancer), Hela (uterine cervix) and A549 (lung adenocarcinoma). Inhibition concentration (IC)(50) was assessed by MTT assay. Cell cycle modifications were determined using dual parameter bromodeoxyuridine and propidium iodide. L-OHP yielded a superior cytotoxicity on HT29 and MCF7 relative to Hela and A549 after treatment, the post-incubations demonstrate that growth inhibition was irreversible for HT29 and Hela cell lines contrary to MCF7 and A549. The main effects of L-OHP are G2/M cell cycle arrest and transient S phase delay. Taken together, L-OHP treatment results on HT29, MCF7 and Hela, are in favor of lengthening the infusion duration to patients during further clinical trials.

Cell cycle arrest and apoptosis induced by oxaliplatin (L-OHP) on four human cancer cell lines.

BACKGROUND: The effects of oxaliplatin (L-OHP) on cell cycle and apoptosis were examined. MATERIALS AND METHODS: The HT29, MCF7, Hela and A549 cell lines were treated and dual parameter flow cytometry BrdU/PI was then performed to monitor cell cycle modifications. Annexin V-FITC and PI probes were used for the detection of apoptosis. RESULTS: The cells were treated for 3 h followed or not by 24 and 72 h of post-incubation. No changes were observed after 3 h of treatment, while after 24 h of post-incubation, all the cells except A549 were predominantly accumulated in the G2/M-phase; this accumulation was time-dependent. Apoptosis induction was late and moderate and was observed only after 12 h of treatment and 24 h of post-incubation. CONCLUSION: L-OHP induced cell cycle arrest and moderate apoptosis; these two activities were time-dependent. These findings warrant further investigation into the potential antitumour activity of L-OHP in MCF7 and Hela cells and in lengthening the infusion duration in order to achieve the acquired cellular modifications.

Change of conformation of the DNA-binding domain of p53 is the only key element for binding of and interference with p73.

Xenopus p53 has biological and biochemical properties similar to those of human p53, except for optimal temperature. The frog protein is fully active at 30 degrees C and inactive at 37 degrees C, leading to a temperature-sensitive behavior similar to that of the human mutant p53Ala(143) and the murine mutant p53Val(135). Using hybrid proteins between human and Xenopus expressed from artificial p53 minigenes, we have been able to demonstrate that change of conformation of the DNA-binding domain is the major determinant of this heat sensitivity. It has been reported that some human tumor-derived p53 mutants can engage in a physical association with p73, thus inhibiting its transactivating properties. The mechanism of this association remains to be elucidated. The nature of the mutant p53 that can engage in this association also remains controversial. Using the unique opportunity of the temperature sensitivity of Xenopus p53, we demonstrate that binding of and interference with p73 require a change of conformation in the p53 protein. This interaction occurs through the DNA-binding domain of p53 only when it is in a denatured state. These results reinforce the notion that mutant p53 with a conformational change can act as a down-regulator of the p73 pathway in human cancer and could confer a selective advantage to the tumor.

TNFalpha potentiates 2-methoxyestradiol-induced mitochondrial death pathway.

Ewing sarcoma cells are resistant to TNFalpha-induced cell death and this resistance results from the activation of the transcription factor NF-kappaB. Here, we investigated whether NF-kappaB activation interferes with 2-Me-induced cell death signaling in Ewing sarcoma cells and we examined the effect of treatment of these cells with 2-Me either alone or in combination with TNFalpha. Our results show that TNFa cooperates with 2-Me to induce apoptosis in Ewing tumor cells through mitochondrial cell death signaling. These results suggest that the use of TNFalpha in combination with 2-Me may be beneficial for Ewing tumor treatment.

Targeted expression of oncogenic K-ras in intestinal epithelium causes spontaneous tumorigenesis in mice.

BACKGROUND & AIMS: Ras oncoproteins are mutated in about 50% of human colorectal cancers, but their precise role in tumor initiation or progression is still unclear. METHODS: This study presents transgenic mice that express K-ras(V12G), the most frequent oncogenic mutation in human tumors, under control of the murine villin promoter in epithelial cells of the large and small intestine. RESULTS: More than 80% of the transgenic animals displayed single or multiple intestinal lesions, ranging from aberrant crypt foci (ACF) to invasive adenocarcinomas. Expression of K-ras(V12G) caused activation of the MAP kinase cascade, and the tumors were frequently characterized by deregulated cellular proliferation. Unexpectedly, we obtained no evidence of inactivating mutations of the tumor suppressor gene Apc, the "gatekeeper" in colonic epithelial proliferation. However, spontaneous mutation of the tumor-suppressor gene p53, a frequent feature in the human disease, was found in 3 of 7 tumors that were tested. CONCLUSIONS: This animal model recapitulates the stages of tumor progression as well as a part of the genetic alterations found in human colorectal cancer. Furthermore, it indicates that activation of K-ras in concert with mutations in p53 may constitute a route to digestive tumor formation and growth, underlining the fact that the pathway to intestinal cancer is not necessarily a single road.

gamma-Glutamyl transpeptidase expression in Ewing's sarcoma cells: up-regulation by interferons.

The genetic hallmark of Ewing's sarcoma family of tumours (ET) is the presence of the translocation t(11;22)(q24;q12), which creates the ET fusion gene, leading to cellular transformation. Five human gamma-glutamyl transpeptidase (gamma-GT) genes are located near the chromosomal translocation in ET. gamma-GT is a major enzyme involved in glutathione homoeostasis. Five human cell lines representative of primary or metastatic tumours were investigated to study whether gamma-GT alterations could occur at the chromosomal breaks and rearrangements in ET. As shown by enzymic assays and FACS analyses, all ET cell lines consistently expressed a functional gamma-GT which however did not discriminate steps of ET progression. As shown previously [Sancéau, Hiscott, Delattre and Wietzerbin (2000) Oncogene 19, 3372-3383], ET cells respond to the antiproliferative effects of interferons (IFNs) type I (alpha and beta) and to a much less degree to IFN type II (gamma). IFN-alpha and -beta arrested cells in the S-phase of the cell cycle. We found an enhancement of gamma-GT mRNA species with IFN-alpha and -beta by reverse transcriptase-PCR analyses. This is reflected by up-regulation of gamma-GT protein, which coincides with the increase in gamma-GT-specific enzymic activity. Similarly, IFNs up-regulate the levels of gamma-GT in another IFN-responsive B cell line. Whether this up-regulation of gamma-GT by IFNs is of physiological relevance to cell behaviour remains to be studied.

Interactions between human monocytes and fibronectin are suppressed by interferons beta and gamma, but not alpha: correlation with Rho-paxillin signaling.

Modulation of the adhesive responses of monocytic cells may reflect their motility at sites of diseased tissues (inflammation, tumors). Integrins alpha5beta1 mediate fibronectin (Fn)-dependent adhesion of human monocytes and their precursors. The effect of type I IFNs (alpha, beta) and type II IFN (gamma) was assessed on the adhesive capacities of promonocytic U937 cells and monocytes. IFN-beta and IFN-gamma abrogated monocytic cell adhesion to Fn, but such impaired cell attachment was not due to altered levels of alpha5beta1 integrins. In contrast, IFN-alpha did not affect cell adhesion to Fn. Participation of cytoskeleton assembly in IFN-mediated cell detachment was evaluated. Activation of RhoA activity with lysophosphatidic acid (LPA) increased 2-fold the adhesion of monocytic cells to Fn in a alpha5beta1-mediated fashion, and IFN-gamma treatment reversed the enhancing effect of LPA. Moreover, U937 cells and monocytes dominantly expressed the 44-46 kDa paxillin forms and IFN-beta and IFN-gamma led to the accumulation of 66-70 kDa paxillin forms. These results indicate that IFN-mediated loss of monocyte adhesion to Fn is associated with changes in the cytoskeleton associated proteins paxillin and Rho.

Gamma-glutamyl transpeptidase activity mediates NF-kappaB activation through lipid peroxidation in human leukemia U937 cells.

Gamma-glutamyl transpeptidase (GGT) is a key enzyme in the catabolism of glutathione (GSH). Recently, it has been reported that the extracellular cleavage of GSH by GGT induced the production of reactive oxygen species (ROS), suggesting that GGT plays a pro-oxidant role. In the present study, we investigated the nature of the oxidative stress generate by glutathione and GGT and the possibility that this stress affects the activity of NF-kappaB a prototypical oxidant-stress-responsive transcription factor. We found that, in the presence of iron, a natural substrate of GGT, glutathione induces lipid peroxidation in U937 cells. This induction depends on GGT activity as it is prevented by the Serine/Borate complex, a GGT inhibitor. We found that y-glutamyl transpeptidase activity induces NF-kappaB DNA binding activity, an effect which is significantly reduced by the addition of GGT inhibitors (Serine/Borate complex and Acivicin). Moreover, we show that lipid peroxidation is involved in GGT-dependent NF-kappaB activation since vitamin E, which completely inhibits GGT-induced generation of lipid peroxides, prevents the GGT-dependent NF-kappaB activation. Finally, inhibition of GGT by either the Serine/Borate complex or by Acivicin resulted in cell apoptosis. This finding suggests that GGT-mediated NF-kappaB activation plays a role in the control of apoptosis in U937 cells.

Peripheral benzodiazepine receptor ligands reverse apoptosis resistance of cancer cells in vitro and in vivo.

The mitochondrial peripheral benzodiazepine receptor (mPBR) is involved in a functional structure designated as the permeability transition pore, which controls apoptosis. Binding of Fas/APO-1/CD95 triggers a prototypic apoptosis-inducing pathway. Using four different human tumor cell lines (T-cell Jurkat, neuroblastoma SHEP, osteosarcoma 143N2, and glioblastoma SNB79 cell lines), all of which express CD95 and mPBR, we investigated the potential role of mPBR ligands in CD95-induced apoptosis. We show that, in vitro, the three mPBR ligands tested (RO5-4864, PK11195, and diazepam) enhanced apoptosis induced by anti-CD95 antibody in Jurkat cells, as demonstrated by mitochondrial transmembrane potential drop and DNA fragmentation. In contrast, RO5-4864, but not PK11195 or diazepam, enhanced anti-CD95 apoptosis in all other cell lines. These effects were obtained in Bcl-2-overexpressing SHEP cell lines, but not in Bcl-X(L) SHEP cell lines. Enhancement of anti-CD95 antibody-induced apoptosis by RO5-4864 was characterized by an increased mitochondrial release of cytochrome c and Smac/DIABLO proteins and an enhanced activation of caspases 9 and 3, suggesting a mitochondrion-dependent mechanism. Preincubation of cells with the different mPBR ligands or anti-CD95 did not affect the levels of expression of either mPBR or CD95. In vivo, we found that the RO5-4864 mPBR ligand significantly increased the growth inhibition induced by two chemotherapeutic agents, etoposide and ifosfamide, using two human small cell lung cancers xenografted into nude mice. Peripheral benzodiazepine receptor ligands may therefore act as chemosensitizing agents for the treatment of human neoplasms.